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Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

Müller MA, van der Hoek L, Voss D, Bader O, Lehmann D, Schulz AR, Kallies S, Suliman T, Fielding BC, Drosten C, Niedrig M - Virol. J. (2010)

Bottom Line: Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein.We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Bonn Medical Centre, Bonn, Germany.

ABSTRACT

Background: Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses.

Results: In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.

Conclusions: This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

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N-glycosylation of hCoV-NL63 ORF 3 protein. HCoV-NL63 ORF 3 protein with and without a C-terminal V5 tag, and with an N16Q exchange in the tagged version was in-vitro translated in presence of 35S-methionine. SARS-CoV M protein without a tag was translated in the same system as a control. Proteins were digested with endoglycosidase (Endo H) as indicated below each lane, subjected to SDS-PAGE, and visualized. Note the removal of the bands of increased molecular weight for the control and ORF 3 proteins, but not for the ORF 3 protein with an amino acid exchange at the hypothetical N-glycosylation site. Note also that extent of size reduction for the SARS-CoV M protein, which is known to have one N-terminal N-glycosylation site, is the same for the NL63 ORF 3 protein.
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Figure 7: N-glycosylation of hCoV-NL63 ORF 3 protein. HCoV-NL63 ORF 3 protein with and without a C-terminal V5 tag, and with an N16Q exchange in the tagged version was in-vitro translated in presence of 35S-methionine. SARS-CoV M protein without a tag was translated in the same system as a control. Proteins were digested with endoglycosidase (Endo H) as indicated below each lane, subjected to SDS-PAGE, and visualized. Note the removal of the bands of increased molecular weight for the control and ORF 3 proteins, but not for the ORF 3 protein with an amino acid exchange at the hypothetical N-glycosylation site. Note also that extent of size reduction for the SARS-CoV M protein, which is known to have one N-terminal N-glycosylation site, is the same for the NL63 ORF 3 protein.

Mentions: According to in-silico predictions the ORF 3 protein contained three putative N-glycosylation sites at positions 16, 119 and 126 (Figure 1B, Table 1). Only position 16 was considered a possible N-glycosylation target, as the other two positions would be located within the membrane. In a vector expressing ORF 3 protein with a C-terminal V5 tag, asparagine (N) at position 16 was changed into glutamine (Q). In-vitro translated 35S-radiolabelled proteins with and without the exchange were treated or not treated with endoglycosidase H prior to SDS-PAGE analysis. SARS-CoV M protein served as the control because it had been shown previously to be N-glycosylated exclusively at position four [34]. In-vitro translated NL63 protein ORF 3 with and without the V5 tag, but not the same protein with an N16Q exchange, showed a second band of increased molecular weight in SDS-PAGE that disappeared upon endoglycosidase H treatment (Figure 7). In the same way as for SARS-CoV M-protein, deglycosylation did not change the apparent molecular weight of the lower band, verifying absence of any further active glycosylation sites.


Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

Müller MA, van der Hoek L, Voss D, Bader O, Lehmann D, Schulz AR, Kallies S, Suliman T, Fielding BC, Drosten C, Niedrig M - Virol. J. (2010)

N-glycosylation of hCoV-NL63 ORF 3 protein. HCoV-NL63 ORF 3 protein with and without a C-terminal V5 tag, and with an N16Q exchange in the tagged version was in-vitro translated in presence of 35S-methionine. SARS-CoV M protein without a tag was translated in the same system as a control. Proteins were digested with endoglycosidase (Endo H) as indicated below each lane, subjected to SDS-PAGE, and visualized. Note the removal of the bands of increased molecular weight for the control and ORF 3 proteins, but not for the ORF 3 protein with an amino acid exchange at the hypothetical N-glycosylation site. Note also that extent of size reduction for the SARS-CoV M protein, which is known to have one N-terminal N-glycosylation site, is the same for the NL63 ORF 3 protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2819038&req=5

Figure 7: N-glycosylation of hCoV-NL63 ORF 3 protein. HCoV-NL63 ORF 3 protein with and without a C-terminal V5 tag, and with an N16Q exchange in the tagged version was in-vitro translated in presence of 35S-methionine. SARS-CoV M protein without a tag was translated in the same system as a control. Proteins were digested with endoglycosidase (Endo H) as indicated below each lane, subjected to SDS-PAGE, and visualized. Note the removal of the bands of increased molecular weight for the control and ORF 3 proteins, but not for the ORF 3 protein with an amino acid exchange at the hypothetical N-glycosylation site. Note also that extent of size reduction for the SARS-CoV M protein, which is known to have one N-terminal N-glycosylation site, is the same for the NL63 ORF 3 protein.
Mentions: According to in-silico predictions the ORF 3 protein contained three putative N-glycosylation sites at positions 16, 119 and 126 (Figure 1B, Table 1). Only position 16 was considered a possible N-glycosylation target, as the other two positions would be located within the membrane. In a vector expressing ORF 3 protein with a C-terminal V5 tag, asparagine (N) at position 16 was changed into glutamine (Q). In-vitro translated 35S-radiolabelled proteins with and without the exchange were treated or not treated with endoglycosidase H prior to SDS-PAGE analysis. SARS-CoV M protein served as the control because it had been shown previously to be N-glycosylated exclusively at position four [34]. In-vitro translated NL63 protein ORF 3 with and without the V5 tag, but not the same protein with an N16Q exchange, showed a second band of increased molecular weight in SDS-PAGE that disappeared upon endoglycosidase H treatment (Figure 7). In the same way as for SARS-CoV M-protein, deglycosylation did not change the apparent molecular weight of the lower band, verifying absence of any further active glycosylation sites.

Bottom Line: Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein.We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Bonn Medical Centre, Bonn, Germany.

ABSTRACT

Background: Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses.

Results: In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.

Conclusions: This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

Show MeSH
Related in: MedlinePlus